1. Field of the Invention
This invention relates to a family of watersoluble esters and to their use as coupling agents. As coupling agents, the esters react with amine functionalities and thus can couple to biological materials which contain amine groups such as amine-containing proteins, polypeptides. peptides and the like. The coupling agents include a range of reagent moieties including labels and functionalities capable of entering into additional coupling reactions so as to join the amine-containing materials to one another and also to link these materials to supports as matrices, to labels, to haptens or to the like. The coupling process has the special advantage that it gives rise to a very easily monitored leaving group so that the degree of reaction is precisely monitorable and thus precisely controllable and also imparts a pronounced water solubility to the coupling agents.
2. Description of Related Disclosures
In the field of biochemistry it is often useful to cross-link species or to couple different species together so as to alter or improve their properties. For example, although short peptides generally function poorly as immunogens, a vigorous immune response may be elicited by coupling these peptides to protein carriers. Also by way of example, in general, haptens (antigens), biotin, drug derivatives or peptides may be covalently bonded to proteins or natural or synthetic polypeptides for use as immunogens or recognition tags, or to enzymes for use in homogeneous enzyme immunoassays or to other label moieties for use in specific binding assays. Similarly, antibodies may be coupled to toxins for use as immunotoxins, or to enzymes for use in enzyme immunoassays. Such cross-couplings of two generally polyfunctional molecules at defined reaction loci present a considerable challenge.
In these coupling reactions, it is often extremely important to be able to monitor the extent of coupling so as to be able to control it. Some cases, such as conjugating enzymes to antibodies, demand a one-to-one correspondence between the two species being coupled. Other procedures, such as immunogen production, require multiple couplings between many haptens and the carrier protein. The ability to monitor and control the coupling reactions involved in these processes is extremely valuable.
Coupling agents can introduce a reagent compound such as a label into the coupled complex. Alternatively they can provide a site for further coupling, that is be bifunctional. They can be homobifunctional, that is having two identical active groups or two groups which react with identical functionalities. They also can be heterobifunctional so as to react with one functionality on one species and with a different functionality on a second species. Typical homobifunctional agents employed heretofore include bis-isocyanates, bis-imidoesters. bis-diazotized benzidine. glutaraldehyde, bis-anhydrides, diphenyl azides, dimaleimides, and active diesters with carbamyl or thiocarbamyl groups, including the nitrogen and thio analogs thereof, as described in U.S. Pat. Nos. 4,334,069; 4,323,647; and 4,046,636. These agents have disadvantages because they cause intramolecular cross-linking and self-condensation.
Esters of N-hydroxysuccinimide are commonly used as heterobifunctional coupling agents. Examples of these materials include the m-maleimidobenzoyl-N-hydroxysuccinimide ester, as described in U.S. Pat. Nos. 4,253,996 and 4,214,048 and by T. Kitagawa, et al., J Biochem 79:233-236 (1976); the N-(4-carboxycyclohexylmethyl)maleimido-N-hydroxysuccinimide ester, as described by S. Yoshitake, et al., Eur J Biochem 101:395-399 (1979); and the iodoacetyl-N-hydroxysuccinimide ester, as described by E. Rector. et al., J. Immunol Methods 24:321-336 (1978). An additional reagent of this type is N-succinimidyl-3- (2-pyridyldithio)propionate as described by J. Carlsson. et al., Biochem J 173:72-737 (1978) and D. pain, et al., J Immunol Methods 40:219-230 (1981). These prior agents are at best only sparingly soluble in water. In use, they must be dissolved in organic solvents, generally dimethylformamide, and this solution is then added slowly to aqueous solutions containing the species to be coupled. Under such conditions, control of the final concentration of the coupling agent, and thus of the rate and extent of reaction, is quite difficult. Because of this lack of control over the reaction, even when a coupling reaction is successful, it is typically very difficult to reproduce. Moreover, high concentrations of the organic solvent can interfere with the properties of many biologically important materials. For example such solvents can denature or precipitate out proteins.
In an attempt to improve the solubility of the esters, sulfo-N-hydroxysuccininmide have been used as coupling agents. While these esters do exhibit improved solubility in water, they do not lend themselves to processes where monitoring and close control are called for. This is because their leaving groups have low extinction coefficients and the wavelength at which their maximum absorbance occurs is 259 nm, which is close to the 280 nm wavelength region in which most proteins exhibit their maximum absorbance. Therefore, monitorability of the coupling reaction by measuring the absorbance of the detached leaving groups is poor because the proteins establish a high background against which only large changes can be detected. Such conditions do not allow one to detect, for example, one-to-one coupling between immunodiagnostic enzymes and antibodies.
Several o-nitro-p-sulfonic acid-phenyl esters (i.e. esters of 4-hydroxy-3-nitrobenzene sulfonic acid) have been described as coupling agents for amino acids. These materials are highly water soluble. For example, Klausner, et al., in peptides, Proceedings of the Fifth American Peptide Symposium, ed. by M. Goodman, et al. (New York: John Wiley and Sons, 1977). p. 536-538, discloses synthesis of peptides in water using 4-hydroxy-3-nitro-benzene sulfonic acid sodium salt (HNSA) esters of N-protected amino acids. Gershkovich, et al., in Bioorganicheskaya Khimiva 4:1129 (1978). Bioorganicheskaya Khimiya 5:1125 (1979) and Bioorqanicheskaya Khimiya 8:1486-1489 (1982) have similar teachings.
P. Bhatnaqar, et al.. in Peptides:Synthesis-Structure-Function, ed. by D. Rich, et al., (Rockford: Pierce Chemical Company, 1981). pp. 97-100, discloses that the N-2,4-dinitrophenyl-6-aminocaproic acid ester of HNSA is more efficient than the same ester of N-hydroxysuccinimide in conjugating to bovine serum albumin at pH 8.5. In addition, cells treated with biotin-HNSA exhibited a much larger increase in fluorescence than cells treated with biotin-p-nitrophenyl ester, when both were reacted with fluoresceinated avidin.
A special problem which comes up when the coupling involves proteins and peptides as the amine-containing material being coupled is the poor yields obtained. Gershkovich. et al., in Biooroanicheskaya Khimiva 5:1125 (1979) teaches that the rate of aminolysis by glycine (which contains only an alpha-amino group) at pH 7.4 using N-benzyloxycarbonyl-gly-HNSA (Z-gly-HNSA) ester is considerably higher than its hydrolysis rate. However, one of the most favored sites on a protein for chemical modification is not the .alpha.-amino group, but rather the .epsilon.-amino group of lysines because these latter groups are normally on the surface of the protein structure. The .epsilon.-amino group is also favored because there is usually a plurality of .epsilon.-amino groups but only one .alpha.-amino group on a protein or on a protein chain; thus, modification of .epsilon.-amino sites enables multiple functionalization of any given protein. The pK.sub.a of .alpha.-amines is lower than the pK.sub.a of .epsilon.-amine groups of lysine. The pK.sub.a of these latter groups ranges between 8.0 and 10.5 for most proteins, and only the deprotonated amine is sufficiently nucleophilic to attack active esters. Therefore, modification of protein amino groups with esters is normally performed at high pH's--e.g., 8.5, to deprotonate the amine. At such elevated pH's the high concentration of hydroxide ion results in extensive hydrolysis of the ester. This unwanted side reaction generally competes very favorably with the desired amide-forming reaction.
Accordingly, there is a need in the biochemical field for a coupling agent which is not only soluble in water but also can be precisely monitored to control the extent of the coupling reaction and, at the proper pH conditions, will result in high yields of the desired amide product.